The role of hydrogen sulfide in the central nervous system implications in the treatment of alzheimers disease

47 3 Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway .... 53 3.3.2 The involvement of cA

THE ROLE OF HYDROGEN SULFIDE IN THE CENTRAL NERVOUS SYSTEM: IMPLICATIONS IN TREATMENT OF ALZHEIMER’S

DISEASE

BHUSHAN VIJAY NAGPURE

NATIONAL UNIVERSITY OF SINGAPORE

2014

THE ROLE OF HYDROGEN SULFIDE IN THE CENTRAL NERVOUS SYSTEM: IMPLICATIONS IN TREATMENT OF ALZHEIMER’S

DISEASE

BHUSHAN VIJAY NAGPURE

(M.B.B.S., Maharashtra University of Health Sciences, India)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2014

DECLARATION

I hereby declare that this thesis is my original work and it has been written by

me in its entirety I have duly acknowledged all the sources of information that

have been used in the thesis

This thesis has also not been submitted for any degree in any university

previously

Bhushan Vijay Nagpure

21st July 2014

ACKNOWEDGEMENTS

First of all, I would like to express my deepest gratitude to my supervisor, A/P Bian Jinsong, for providing me the opportunity to continue with my graduate studies I appreciate his scientific advices during the course

of study I also want to thank him for his support and encouragement throughout my journey of PhD

Sincere appreciation to the lab technologist, Ms Shoon Mei Leng, for assisting with laboratory matters Many thanks to my fellow past and present colleagues at A/P Bian Jinsong’s laboratory, Wu Zhiyuan, Hua Fei, Cao Xu, Xie Li, Tiong Chi Xin, Li Guang, Liu Yanying, Liu Yitong, Lu Ming and Hu Lifang for their insightful discussion, technical advice and help in one way or another

I would also extend my gratitude to Yong Loo Lin School of Medicine, National University of Singapore for offering me scholarship and providing

me the opportunity to pursue higher studies in Singapore

Finally, I would also like to convey my greatest gratitude to my parents, my brother and my wife for their continuous love, encouragement and support in past few years

TABLE OF CONTENTS

ACKNOWEDGEMENTS II TABLE OF CONTENTS III SUMMARY VII LIST OF TABLES X LIST OF FIGURES XI LIST OF ABBREVEATIONS XII LIST OF PUBLICATIONS XIV

1 Introduction and Literature Review 1

1.1 The trio of gasotransmitters 2

1.1.1 H2S- The ‘third’ gasotransmitter 2

1.1.1.1 Physical and chemical properties of H2S 3

1.1.1.2 Toxicity of H2S 4

1.1.1.3 Biosynthesis of H2S 6

1.1.1.4 Storage and metabolism of H2S 8

1.1.1.5 Biological role of H2S in CNS 9

1.2 Alzheimer’s disease 16

1.2.1 History 16

1.2.2 Epidemiology 17

1.2.3 Risk factors 19

1.2.4 Pathology 20

1.2.4.1 Amyloid β 20

1.2.4.2 Tau 21

1.2.4.3 Studies done on animal models and AD patients 22

1.2.4.4 Other etiopathological hypotheses 23

1.2.5 ATP and its metabolites in AD 26

1.2.6 Diagnosis and clinical symptoms of AD 28

1.2.6.1 Classification and Diagnostic criteria 28

1.2.6.2 Clinical symptoms and the course of the illness 29

1.2.7 Pharmacotherapy 31

1.3 Objectives 34

2 Materials and methods 36

2.1 Chemicals 37

2.2 Cell Culture and Treatments 37

2.3 Constructs and Mutagenesis 39

2.4 Preparation of Primary Astrocyte Culture 40

2.5 Cell Viability Assay 40

2.6 Intracellular cAMP Assay 41

2.7 Cell Fractionation and Adenylyl Cyclase (AC) Activity Assay 41

2.8 γ-secretase (Fluorogenic Substrate) Assay 42

2.9 ELISA for Aβ42 42

2.10 Reactive Oxygen Species (ROS) Measurement 43

2.11 Measurement of Nitric Oxide 43

2.12 ELISA for TNF-α and IL-Iβ 43

2.13 DNA binding activity assay 43

2.14 Cathepsin S activity assay 44

2.15 S-sulfhydration assay (modified biotin switch) 44

2.16 Glutamate uptake assay 45

2.17 Reverse Transcription-PCR 45

2.18 Western Blot Assay 47

2.19 Statistical Analysis 47

3 Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway 49

3.1 Introduction 50

3.2 Materials and Methods 52

3.2.1 Chemicals 52

3.2.2 Cell Culture and Treatments 52

3.2.3 Cell Viability Assay 52

3.2.4 Intracellular cAMP Assay 52

3.2.5 Cell Fractionation and Adenylyl Cyclase (AC) Activity Assay 52

3.2.6 γ-secretase (Fluorogenic Substrate) Assay 52

3.2.7 ELISA for Aβ42 52

3.2.8 Reverse Transcription-PCR 52

3.2.9 Western Blot Assay 52

3.2.10 Statistical Analysis 53

3.3 Results 53

3.3.1 NaHS attenuates adenosine A2A receptor agonist stimulated Aβ42 production 53

3.3.2 The involvement of cAMP/PKA/CREB pathway in the inhibitory effect of NaHS on HENECA-stimulated Aβ42 production 56

3.3.3 NaHS targets activated AC, not A2A receptors 59

3.3.4 NaHS inhibits APP production and maturation 62

3.3.5 NaHS attenuates γ–secretase activity, not of β–secretase 64

3.3.6 Effect of NaHS on expression of presenilins 67

3.4 Discussion 68

4 Hydrogen sulfide repairs impaired glutamate uptake in A2A adenosine receptor agonist-stimulated primary astrocytes 77

4.1 Introduction 78

4.2 Material and Methods 79

4.2.1 Chemicals 79

4.2.2 Primary astrocyte culture 80

4.2.3 Intracellular cAMP Assay 80

4.2.4 Glutamate uptake assay 80

4.2.5 Statistical analysis 80

4.3 Results 80

4.3.1 The effect of NaHS on glutamate uptake in A2A adenosine receptor agonist stimulated astrocytes 81

4.3.2 The involvement of cAMP signaling pathway in the protective effect of NaHS on HENECA-stimulated astrocytes 82

4.4 Discussion 83

5 Hydrogen sulfide inhibits the Aβ synthesis and neuroinflammation in extracellular ATP-stimulated BV-2 microglia cells via inhibition of NF-κB , STAT3 and cathepsin S activation 86

5.1 Introduction 87

5.2 Materials and Methods 90

5.2.1 Chemicals 90

5.2.2 Cell Culture and Treatments 90

5.2.3 Constructs and mutagenesis 90

5.2.4 Cell Viability Assay 90

5.2.5 Intracellular cAMP Assay 90

5.2.6 γ-secretase (Fluorogenic Substrate) Assay 90

5.2.7 ELISA for Aβ42 90

5.2.8 Reactive Oxygen Species (ROS) Measurement 90

5.2.9 Nitric oxide (NO) Measurement 90

5.2.10 DNA binding activity assay 91

5.2.11 Cathepsin S activity assay 91

5.2.12 S-sulfhydration assay 91

5.2.13 Western Blot Assay 91

5.2.14 Statistical Analysis 91

5.3 Results 91

5.3.1 Effect of NaHS on ATP-induced oxidative stress and inflammation

in BV-2 microglial cells 91 5.3.2 Effect of NaHS on ATP-induced iNOS and COX-2 expression in microglial cells 94 5.3.3 Effect of NaHS on DNA binding and transcriptional activities of NF-κB in ATP-stimulated microglial cells 96 5.3.4 Effect of NaHS on ATP-induced Aβ42 production in microglial cells

98

5.3.5 Effect of NaHS on STAT3 activity in microglial cells 101 5.3.6 Involvement of cathepsin S in the observed effects of NaHS on ATP-induced neuroinflammation and Aβ production 103

5.4 Discussion 107

6 General Discussion, Limitations of study, Future directions and

Conclusion 113 Bibliography 120

SUMMARY

In today’s world, Alzheimer’s disease (AD) is the leading cause of dementia in elderly population across the world It is also the most common neurodegenerative disease With high prevalence and ever growing incidence rate, AD is set to become one of the most crippling diseases in developed and developing countries Currently, only few approved drugs are available for the treatment of AD Majority of them are prescribed to alleviate neuropsychiatric symptoms without targeting underlying pathological mechanism Hence, a lot

of efforts have been put into the development of disease-modifying drug therapy

Amyloidogenesis is one of the main culprits of AD pathology The effect of NaHS, a rapid exogenous hydrogen sulfide (H2S) donor, was first examined in SH-SY5Y cells transfected with amyloid precursor protein (APP) Swedish mutation H2S pretreatment was found to exert an inhibitory effect on Aβ42 synthesis by HENECA (a selective A2A receptor agonist)-stimulated SH-SY5Y cells NaHS also interfered with the maturation process of APP by inhibiting its generation and post-translational modification A further study of the rate limiting steps of Aβ synthesis i.e β- and γ-secretase activities yielded interesting results H2S did not affect the β-secretase activity However, γ-secretase activity measurement and gene expression study of presenilins revealed that H2S directly inhibited γ-secretase Curiously, H2S also abrogated intracellular cAMP levels and phosphorylation of downstream CREB H2S had similar suppressive effects on cAMP and Aβ42 generation caused by specific

AC stimulation A further study showed that HENECA-stimulated AC activity

and gene expression of AC isoforms were preferentially blocked by H2S while exerting its inhibitory action on Aβ synthesis

A2A adenosine receptors are known to modulate glutamate uptake in astroglial cells When incubated with HENECA, protein expression of GLAST glutamate transporter and glutamate uptake were significantly inhibited in astrocytes The pretreatment with NaHS significantly improved the impaired glutamate uptake and expression of GLAST glutamate transporter Being positively linked to AC, stimulation of A2A receptors by HENECA resulted into the increase in intracellular cAMP levels Similar to the first part of the studies, NaHS inhibited the cAMP production in astrocytes These data suggest that H2S -inhibited cAMP production was probably responsible for its regenerative effect on glutamate uptake and restoration of GLAST

Several line of evidences show that severe neuroinflammation leads to amyloidogenesis in the CNS By the detailed analysis of generation of many inflammatory parameters, it was found that H2S pretreatment suppressed extracellular ATP-induced severe neuroinflammation in immortalized BV-2 cells While exerting its anti-inflammatory effect, H2S also imparted the inhibitory effect on Aβ synthesis in microglia NF-κB and STAT3 are responsible for transcription of many inflammatory genes The activation of both the transcription factors was blocked by H2S Cathepsin S, which was found to be situated downstream to STAT3 in the current study, was involved

in β-secretase cleavage of APP and NF-κB activation We found that H2S sulfhydrated Cathepsin S and inhibited its expression and activity in ATP-stimulated BV-2 cells

s-In conclusion, the present study demonstrated that H2S is a potent neuroprotective agent against AD pathology H2S therapy has a potential to be

an effective and promising therapeutic strategy against AD as it acts on the multiple targets in the pathological process of the disease

LIST OF TABLES

Table 1 Health effects of H 2 S at various approximate exposure levels 6 Table 2 Prevalence and incidence of dementia in developed and developing regions 19 Table 3 Evaluation of dementia 31

LIST OF FIGURES

Figure 3.1 Effects of NaHS on Aβ42 synthesis and cell viability in APPswe

transfected SH-SY5Y cells 55

Figure 3.2 The involvement of cAMP signaling pathway in the observed effects of NaHS on Aβ42 production 59

Figure 3.3 Effect of NaHS on genes expression of neuron-specific AC isoforms and AC activity 62

Figure 3.4 Inhibitory effects of NaHS on APP production and maturation 64

Figure 3.5 Different effect of NaHS on activities of β- and γ-secretases 66

Figure 3.6 NaHS inhibits mRNA expressions of presenilins 1 and 2 68

Figure 3.7 Schematic diagram depicting the inhibitory effect of H2S on HENECA induced Aβ production in APPswe transfected SH-SY5Y cells 76

Figure 4.1 Effect of NaHS on GLAST protein expression ………… …….81

Figure 4.2 Effect of NaHS on glutamate uptake……… ………82

Figure 4.3 Effect of NaHS on glutamate uptake in HENECA-stimulated astrocytes involves cAMP signaling pathway……… 83

Figure 5.1: Effect of NaHS on ATP-induced oxidative stress and inflammation in BV-2 microglial cells……… 94

Figure 5.2: Effect of NaHS on protein expression of iNOS and COX-2…….96

Figure 5.3: Effect of NaHS on DNA binding and transcriptional activities of NF-κB……… 98

Figure 5.4: Effect of NaHS on ATP-induced Aβ42 production………100

Figure 5.5: Effect of NaHS on STAT3 activity……….102

Figure 5.6: Involvement of cathepsin S……….106

LIST OF ABBREVEATIONS

Aβ42 β-Amyloid 1-42

AD Alzheimer‘s disease

APP Amyloid precursor protein

ATP Adenosine triphosphate

BACE1 β-site amyloid precursor protein cleaving enzyme 1 cAMP 3'-5'-Cyclic adenosine monophosphate;

CAT Cysteine aminotransferase

ELISA Enzyme linked immunosorbent assay

GABA γ-aminobutyric acid

iNOS Inducible nitric oxide synthase

KATP ATP-sensitive potassium channel

LPS Lipopolysaccharide

LTP Long-term potentiation

MAO Monoamine oxidase

3-MST 3-mercaptopyruvate sulfurtransferase

mitoKATP Mitochondrial KATP channel

NaHS Sodium hydrogen sulfide

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NMDA N-methyl-D-aspartic acid

NO Nitric oxide

NSAIDs Non-steroidal anti-inflammatory drugs

PCR Polymerase chain reaction

PD Parkinson‘s disease

PKA Protein kinase A

ROS Reactive oxygen species

STAT3 Signal transducers and activators of transcription 3

TNF-α Tumor necrosis factor-α

LIST OF PUBLICATIONS

Original Research Papers-

¤ Nagpure BV, Bian JS H2S inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway PLOS One 2014 Feb 11;9(2):e88508 doi: 10.1371/journal.pone.0088508 eCollection 2014

¤ Liu YY, Nagpure BV, Bian JS H2S protects SH-SY5Y neuronal cells against d-galactose induced cell injury by suppression of advanced glycation end products formation and oxidative stress Neurochem Int

2013 Apr;62(5):603-9 doi:10.1016/j.neuint.2012.12.010 Epub 2012 Dec 26

¤ Nagpure BV, Wu Zhiyuan, Bian JS H2S inhibits neuroinflammation and the production of β-amyloid in BV-2 microglia cells by inhibiting STAT3 and Cathepsin S (Ready for submission to Antioxidants & Redox Signaling)

Invited Review Paper-

¤ Nagpure BV, Bian JS Interaction of hydrogen sulfide with nitric oxide in the cardiovascular system (Accepted in Oxidative Medicine and Cellular Longevity)

Invited Book Chapters-

¤ Nagpure BV, Bian JS Brain, learning and memory: Role of H2S in neurodegenerative diseases Chemistry, Biochemistry and Pharmacology of H2S (Submitted and in communication with the editors)

¤ Yang HY, Nagpure BV, Bian JS Opioid dependence and the adenylyl cyclase/cAMP signaling The Neuropathology Of Drug Addictions And Substance Misuse (Submitted and in communication with the editors)

International/ Local Conferences presentations

¤ Nagpure BV, Bian JS Hydrogen Sulfide: A novel agent to protect kidney against hypertensive renal injury Oral Presentation at Second

Scientific meeting, National Kidney Foundation, Singapore (March 2014)

¤ Nagpure BV, Bian JS Hydrogen sulfide attenuates Beta-Amyloid production in a cell model of Alzheimer's disease Poster Presentation

at International Conference of Pharmacology and Drug Development, Singapore (December 2013)

¤ Nagpure BV, Bian JS Neuroprotective effect of hydrogen sulfide: regulation of amyloidosis and inflammation in SH-SY5Y

neuroblastoma and BV-2 microglia cells Oral Presentation at Second European Conference on the Biology of Hydrogen Sulfide, Exeter, UK (Sept 2013)

1 Introduction and Literature Review

1.1 The trio of gasotransmitters

In recent few decades, the scientific community has witnessed the rise of a whole new class of gaseous biological mediators in mammalian cells The size

of this family is growing continually over the years since the seminal discovery of physiological effects of nitric oxide (NO) on blood vessels (Ignarro et al., 1987) With relatively recent recognition of carbon monoxide (CO) and hydrogen sulfide (H2S) as two more gaseous signaling molecules, the term ‘gasotransmitter’ was coined As the name suggests, they are simple gas molecules, which are lipid soluble and hence freely membrane permeable reaching intracellular organelle In contrast to the conventional idea of typical signaling molecules, these gasotransmitters are synthesized endogenously on

‘as and when required’ basis from specific enzymes in highly regulated manner Many studies over decades studies have revealed that these gasotransmitters have specific molecular targets and well defined biological functions at physiological concentrations (Wang, 2002b)

1.1.1 H 2 S- The ‘third’ gasotransmitter

The physiological role of H2S was discovered by a Japanese group of scientists led by Abe and Kimura in 1996 In their pioneering study, the novel neuromodulator role of H2S was transpired (Abe and Kimura, 1996) Since then its possible roles in all body systems were and are being investigated worldwide In mammalian central nervous system (CNS), its prominent effects include modulation of neurotransmission and long-term potentiation (Abe and Kimura, 1996) and induction of neuroprotection (Hu et al., 2011) from myriad

of pathogenic agents In mammalian cardiovascular system (CVS), its protective effects are deeply studied (Polhemus et al., 2014, Liu et al., 2012) The induction of relaxation (Yang et al., 2008) and constriction (Kohn et al., 2012) in various types of blood vessels is also documented (d'Emmanuele di Villa Bianca et al., 2011) The opposite effects of H2S on systemic and localized inflammation have been observed in various mammalian tissues (Whiteman and Winyard, 2011, Hegde and Bhatia, 2011) As more and more physiological and pharmacological implications of H2S are explored, we can say that its whiff has blossomed (Wang, 2012)

1.1.1.1 Physical and chemical properties of H 2 S

At room temperature and ambient pressure, H2S exists in a colorless gaseous form The smell is very pungent with distinctive rotten-egg odour It

is readily water soluble due to its weak acidic nature Its solubility was measured to be 80 mM at 37 °C as equilibrium between H2S, HS- and S2- The acid dissociation constant (pKa) values of the first and second dissociation steps are recorded as 7.0 and >12.0, respectively (Vorobets et al., 2002, Kabil and Banerjee, 2010, Mark et al., 2011) Thus, at physiological pH of 7.4, H2S exists majorly as HS- moiety along with minor presence of free H2S in its dissociated form The minute amounts of sulfide anions (S2-) can also be detected Even with the advent of various methods of H2S measurement, it’s almost impossible to determine the active form of H2S (H2S, HS- or S2-) present in the biological system Hence the all-encompassing term of H2S is now used to refer the total sulfide content present in the solution (i.e H2S +

HS-+ S2-) (Zhao et al., 2014b)

1.1.1.2 Toxicity of H 2 S

H2S is often a lethal environmental and occupational hazard that has a unique pattern of toxicity It is the second most common cause (at 7.7%, after carbon monoxide at 36%) of fatal gas inhalation exposure at the workplaces like oil rigs and urban sewers (Guidotti, 2010) Its exposure-response curve for lethality is steep, thus concentration of inhaled gas is more important compared to the duration of exposure (Prior et al., 1988, Guidotti, 1996) The approximate concentrations (exposure levels) of inhaled H2S for the major toxicological effects are given in table 1 The toxidrome (i.e a set of symptoms and signs associated with a particular poison) of H2S is often considered as one of the most unusual and reliable toxidromes (Milby and Baselt, 1999, Wang, 1989) It is characterized by the ‘knockdown’ (acute central neurotoxicity), pulmonary edema, conjunctivitis and odor perception followed by respiratory paralysis (Guidotti, 2010) Acute toxicity leading to reversible unconsciousness caused by H2S inhalation are called as

‘knockdowns’ (Guidotti, 1996) Although knockdowns can be fatal in the cases of prolonged high-concentration exposure of about 500-1000 ppm (about 15-30 mM), the transient exposure is often reversible and apparently complete functionally (Burnett et al., 1977) Pulmonary edema is a well-recognized effect of acute H2S toxicity As H2S has relatively high solubility,

it penetrates deeply into respiratory track, causing alveolar injury culminating

in acute pulmonary edema (Guidotti, 2010) The conjunctivitis, caused by prolonged low-concentration exposure of about 20 ppm (about 500 uM) (Lambert et al., 2006) is peculiarly associated with reversible chromatic distortion and visual changes These symptoms are sometimes accompanied

by blepherospasm and photophobia (Tansy et al., 1981, Milby and Baselt, 1999) H2S is an odorous gas at low concentration of 0.01-0.3 ppm (about 0.09 mM) As the concentration increases, however, the victims start to experience olfactory fatigue It is a sensory adaptation where the victims get accustomed

to strong odor At around 100 ppm (about 3 mM) concentration, H2S paralyses the olfactory mechanism, preventing perception of any smell This phenomenon removes the primary warning sign of H2S exposure (Ronk and White, 1985, Turner et al., 1990)

Concentration

(ppm)

Effects

0.01-0.3

Minimum concentration detected by human nose (may

differ from person to person)

1-5

Generally tolerated pungent smell, although some people can shows symptoms like nausea, mild lacrimation and possible heavy-headedness

10 Threshold for anaerobic metabolism in normal person

100 Eye and lung irritation; olfactory paralysis,

disappearance of character pungent odor

150-200 Severe eye and lung irritation, sense of smell paralyzed 250-500 Long term exposure might lead to pulmonary edema

500

Serious damage to eyes, severe lung irritation, knockdown and death within 4-8 hours, amnesia for

period of exposure

1000 Immediate cessation of breathing; instant collapse

Table 1 Health effects of H 2 S at various approximate exposure levels Data is reproduced from (Guidotti, 1996, Guidotti, 2010) with some modifications

1.1.1.3 Biosynthesis of H 2 S

In mammalian tissues, H2S is biosynthesized from amino acid cysteine (Cys) and homocysteine (Hcy), which are recognized as the principle substrates for its endogenous production They are acted upon by three different enzymes, namely cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST) (Hu et al., 2011) Expressions of these enzymes are variable in different tissues The study of this variation is important as the modulation of endogenous production of H2S can be achieved by targeting each enzyme separately or concurrently

A pyridoxal-5’-phosphate (PLP)-dependent enzyme, CBS, initiates the trans-sulfuration pathway by catalyzing β-replacement of serine by Hcy to generate cystathionine and water Furthermore, serine replacement by cysteine

as a substrate results into production of cystathionine and H2S Besides above mentioned trans-sulfuration pathway, CBS also catalyzes condensation

reactions between two molecules of Cys and β-replacement of Cys by water to produces H2S (Kabil and Banerjee, 2014) The reaction replacing Hcy by Cys yields maximum generation of H2S in vitro (Singh et al., 2009) CBS is found

to be primarily expressed in various regions of the human brain (Abe and Kimura, 1996)

CSE is yet another PLP-dependent enzyme, which mediates a reaction between thiocysteine and a thiol compound R-SH to generate H2S (Kimura, 2011) The substrate thiocysteine is generated from L-cystine which in turn is produced by two L-cysteine molecules (Yamanishi and Tuboi, 1981) Expression of CSE is rather widely distributed among peripheral tissues including liver, pancreas, uterus and intestine (Kimura, 2011) It is the main H2S-generating enzyme in the cardiovascular system (Zhao et al., 2001, Bian

et al., 2006) CSE was detected in relatively large amounts in the myocardium (Geng et al., 2004), endothelial cells (ECs) (Yang et al., 2008), and smooth muscle cells (Zhao et al., 2001)

The third enzyme, 3-mercaptopyruvate sulfotransferase (3-MST), was identified in the neurons The research group detected the significant presence

of H2S in the brain homogenate preparation of CBS-/- mice (Shibuya et al., 2009) Kimura further observed that 3-MST acts together with cysteine aminotransferase (CAT) to generate H2S from Cys in the presence of α-ketoglutarate (Kimura et al., 2010) However, it is suggested that 3-MST is unable to produce H2S in normal physiological conditions as they exert their activities at higher alkaline pH level Furthermore, it requires endogenous reducing substances such as thioredoxin and dihydrolipoic acid (DHLA) for the production of H2S (Kimura, 2014) Aspartate can also act as a substrate for

CAT, competitively binding to it and attenuating H2S synthesis (Guo et al., 2012)

Recently, Shibuya et al discovered the additional pathway for H2S biosynthesis in mammalian cells 3-MST along with D-Amino acid oxidase (DAO) produces H2S from D-Cysteine by the interaction of mitochondria and peroxisomes It was evident that this D-Cysteine dependent pathway operates predominantly in the cerebellum and the kidney The protective effects of D-Cysteine were observed against oxidative stress in cerebellar neurons and against ischaemia-reperfusion injury in the kidney (Shibuya and Kimura, 2013)

1.1.1.4 Storage and metabolism of H 2 S

Although endogenous H2S can be synthesized and released immediately, the storage forms of H2S are also known Acid-labile sulfur is primarily contained in iron-sulfur center of mitochondrial enzymes and can release H2S only in acidic pH of 5.4 Due to higher instability of iron-sulfur complexes, the release of H2S is readily achieved Bound sulfane sulfur, which

is localized in cytoplasm, consists of divalent sulfur bond (e.g persulfide form) It releases H2S under reducing conditions of pH 8.4 (Ishigami et al., 2009) It is possible that H2S produced by 3-MST/CAT enzymatic pathway is stored in the bound sulfane sulfur form The decreased amount of bound sulfane sulfur has been detected in cells without 3-MST/CAT compared to the cells with it (Shibuya et al., 2009)

H2S is catabolized in mammalian cells though various pathways The major mechanism is through its mitochondrial oxidation in different tissues

(Hildebrandt and Grieshaber, 2008) In a reaction catalyzed by quinone oxidoreductase enzyme, H2S is converted into persulfides Persulfides are, in turn, oxidized in sulfite and thiosulfite In physiological normoxic conditions, the thiosulfite is further metabolized into excretable form of sulfate H2S catabolism by quinone oxidoreductase enzyme seems to be universal in mammalian tissues, with possible exception of the brain (Mikami et al., 2011) H2S can also be methylated to produce methane thiol by the action of enzyme thiol-S-methyltransferase Non-mitochondrial heme proteins such as hemoglobin and myoglobin also catabolize intracellular H2S by oxidation (Berzofsky et al., 1971, Stein and Bailey, 2013) To a smaller extent, H2S can also interact with reactive oxygen and nitrogen species It is interesting to know that the presence of oxygen (O2) is very influential factor in deciding the fate of cellular H2S as O2 is capable of spontaneous oxidization of H2S (Stein and Bailey, 2013, van Kampen and Zijlstra, 1983) The intracellular concentration of H2S is firmly kept in low range, owing to the highly efficient nature of above-mentioned mechanisms

1.1.1.5 Biological role of H 2 S in CNS

1.1.1.5.1 Physiological Roles

The protective effects of H2S on various cell types of CNS in various

in vitro experiments and animal models are being widely investigated Oxidative stress caused by overproduction of reactive oxygen species (ROS) is detrimental and one of the etiological factors of many neurodegenerative diseases Kimura et al found that H2S protects primary neurons from oxidative glutamate toxicity (oxytosis) caused by glutamate H2S elevated antioxidant

glutathione levels by enhancing the activity of gamma-glutamylcysteine synthetase and up-regulating cystine transport The upregulation in gamma-glutamylcysteine synthetase activity facilitates the redistribution of GSH into mitochondria, thus protecting cells against oxidative stress damage (Kimura and Kimura, 2004) Later, the same group also discovered that H2S protects immortalized mouse hippocampal cells from oxytosis by activating ATP-dependent K+ (KATP) and Cl- channels, in addition to increasing the levels of glutathione (Kimura et al., 2006) A study conducted by Lu et al demonstrated that H2S protects astrocytes via enhancing glutamate uptake function of glutamate transporter-1 and elevating glutathione (GSH) production This phenomenon prevents excessive accumulation of glutamate in synaptic clefts protecting neurons from excitotoxicity (Lu et al., 2008) Besides these, H2S downregulates peroxynitrite-mediated tyrosine nitration and inactivation of alpha1-antiproteinase inhibiting peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells (Whiteman et al., 2004) Apart from anti-oxidation effects, H2S is also known to possess anti-apoptotic properties conferring neuroprotection Hu and colleagues discovered that H2S inhibits apoptosis induced by rotenone (a toxin used to establish Parkinson’s disease model) by preserving mitochondrial functions in human neuroblastoma cell line (SH-SY5Y) They observed that H2S regulated the mitoKATP channel and thus impeded the apoptosis cascade (prevention of mitochondrial membrane potential (MMP) dissipation, cytochrome c release and caspase-9/3 activation) (Hu et al., 2009) The anti-apoptotic effect was supported by other studies as well Zhang et al found out that H2S attenuated neuronal injury induced by

vascular dementia via inhibiting apoptosis in rats (Zhang et al., 2009) In yet another study, H2S imparted the cytoprotective effect to PC12 cells against amyloid β (25-35)-induced apoptosis (Tang et al., 2008)

One of the most important and widely studied roles of H2S is its action

as neuromodulator by regulating neurotransmission in and between neurons

It particularly stimulates N-methyl-D-Aspartate (NMDA) receptor mediated currents facilitating the induction of long-term potentiation (LTP) and synaptic plasticity Both phenomena are involved in learning and memory H2S increases NMDA receptor sensitivity to glutamate through activation of downstream adenylyl cyclase (AC) and ensuing cAMP/protein kinase A (PKA) pathway (Abe and Kimura, 1996) As discussed earlier, H2S facilitates clearing of excessive glutamate from synaptic clefts, thus maintaining normal inter-neuronal signaling unhampered (Lu et al., 2008)

The effect of H2S on intracellular calcium [Ca2+]i deserves a special mention here [Ca2+]i is critical for normal neuron-glia communication and regulation of synaptic plasticity It has been found that H2S is capable of regulating [Ca2+]i in all important brain cell types; namely neurons (Yong et al., 2010), microglia (Lee et al., 2006b) and astrocytes (Nagai et al., 2004) There are two main mechanisms by which [Ca2+]i is elevated in H2S stimulated cells; one by its release from intracellular calcium store and other

by its influx via calcium channels located on plasma membrane H2S, having multi-targeted actions, stimulate different channels and secondary signaling pathways It activates L-/T-type calcium channels and NMDA receptors on plasma membrane (Lee et al., 2006b) Yong at al discovered that the action of H2S on [Ca2+]i can be suppressed by using inhibitors of PKA, phospholipase C

(PLC) and protein kinase C (PKC), suggesting the role of PKA and PLC/PKC pathways in the regulatory effect of H2S on [Ca2+]i (Yong et al., 2010) Furthermore, in a recent study conducted by Sekiguchi et al demonstrated that the function of Cav3.2 T-type Ca2+ channels (T-channels) is tonically enhanced by endogenous H2S synthesized by CSE in HEK293 cells transfected with Cav3.2 , and that exogenous H2S is capable of enhancing Cav3.2 function when endogenous H2S production by CSE is inhibited (Sekiguchi et al., 2014) In yet another study done previously by the same group suggests the T-type Ca2+ channels are involved in induction of neuritogenesis and expression of high-voltage-activated currents in NG108-15 cells by H2S (Nagasawa et al., 2009)

1.1.1.5.2 Pathological Roles

Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease histopathologically characterized by progressive degeneration of dopaminergic neurons in substantia nigra of midbrain

Many studies indicate that hyperhomocysteinemia (abnormally high level of homocysteine in the blood) is common in the patients of PD (O'Suilleabhain et al., 2004, Zoccolella et al., 2010) Various experiments in

PD animal models detected reduced levels of H2S in substantia nigra and striatum regions of the brain These findings suggest that impaired endogenous production of H2S has a substantial effect on pathogenesis and progression of

PD Furthermore, the administration of exogenous H2S has shown protective effects against underlying pathologic mechanisms The study conducted by

Zhau et al demonstrated that NaHS, a fast H2S donor, protected PC12 cells from cytotoxicity and apoptosis induced by MPP+, the active metabolite of MPTP They found that H2S inhibited the loss of MMP and the accumulation

of intracellular ROS (Yin et al., 2009) Recently, Li et al (Xie et al., 2013) confirmed the initial findings of Calvert and colleagues (Calvert et al., 2009) about upregulation of endogenous antioxidants by H2S via stimulation of nuclear-factor-E2–related (Nrf2)-dependent signaling pathway Previously, it was shown that pretreatment with NaHS can protect human neuroblastoma SH-SY5Y cells against rotenone-induced apoptosis (Hu et al., 2009) and 6-OHDA-induced cell injury (Tiong et al., 2010)

These findings in vitro studies were supported by the observations done in animal models of PD Hu et al found out that the systemic administration of NaHS dramatically reversed the progression of movement dysfunction, loss of tyrosine-hydroxylase (TH) positive neurons in the striatum and the elevated malondialdehyde level in injured striatum caused by 6-OHDA or rotenone (Hu et al., 2010) Inhaled H2S also prevented the MPTP-induced movement disorder and the degeneration of TH-containing neurons

by upregulating heme oxygenase-1 and glutamate-cysteine ligase (Kida et al., 2011) The anti-inflammatory, antioxidant and neuroprotective properties shown by new H2S releasing hybrids are encouraging thus making them ideal candidates for PD treatment ACS 84, a well-known L-DOPA hybrid, has been effective in reducing the release of pro-inflammatory cytokines and NO from stimulated microglia and astrocytes (Lee et al., 2010) Beside relieving from inflammation, H2S releasing L-DOPA hybrids also restore the depleted

dopamine levels by inhibiting mono-amine oxidase B activity (Sparatore et al., 2011)

These findings highlight the potential therapeutic benefit of H2S in PD which can be achieved either by the administration of exogenous H2S or the modulation of endogenous H2S production

Vascular Dementia

Vascular dementia (VD), a heterogeneous group of brain disorders in which cognitive impairment is attributable to cerebrovascular pathologies, is responsible for at least 20% of cases of dementia, being second only to Alzheimer’s disease (Gorelick et al., 2011, Iadecola, 2013) A pioneering study done by Zhang et al suggests that H2S could protect the brain against

VD injury induced by cerebral ischemia reperfusion through inhibiting the apoptosis in the hippocampus They found that NaHS-treated rats had a greater ratio of Bcl-2 (anti-apoptotic) over Bax (pro-apoptotic) with increased Bcl-2 expression and decreased Bax expression in the hippocampus (Zhang et al., 2009) It is generally believed that inflammation (Malaguarnera et al., 2006, Liu et al., 2007), oxidative stress (Liu et al., 2007) and vascular factors (Brown et al., 2007, Stephan and Brayne, 2008) play important roles in the

VD pathology As discussed earlier in this thesis, H2S possesses potent inflammatory (Hu et al., 2007b) and anti-oxidative action (Kimura and Kimura, 2004) It has also been shown that H2S exerts cardioprotective action against myocardial ischemia reperfusion injury (Elrod et al., 2007) Hence, it appears that H2S may protect against VD injury by targeting multiple signaling pathways and events

anti-Ischemic stroke

Ischemic stroke often results into loss of brain functions due to the neuronal damage in the ischemic area (Mestriner et al., 2013) Hippocampus is one of the regions more prone to an ischemic insult (Gordan et al., 2012) Hippocampus plays a very important role in memory retention and spatial navigation Thus, damages to this region cause a significant loss in memory and learning (Wen et al., 2014)

It has been known that exogenous H2S treatment improves myocardial dysfunction related to ischemia/reperfusion injury (Lowicka and Beltowski, 2007) The beneficial effects of H2S were also investigated in CNS models of ischemia An interesting study done on mild focal cerebral ischemia rat model showed that H2S at a low concentration remarkably lessened the injury (Florian et al., 2008) Kimura et al demonstrated that H2S reinstated glutathione (GSH) levels in the fetal brain decreased by ischemia/reperfusion

in utero (Kimura et al., 2010) While studying the effects of H2S on global cerebral ischemia–reperfusion (I/R), Yin and colleagues found out its potent protective effect against a severe cerebral injury through the inhibition of oxidative stress, inflammation and apoptosis (Yin et al., 2013) Recently Wang et al reported that H2S donors protected blood brain barrier integrity in MCAO (middle cerebral artery occlusion) rat model by inhibiting NF-κB and suppressing post-ischemic inflammation-induced Matrix Metalloproteimase-9 (MMP9) and Nicotinamide adenine dinucleotide phosphate oxidase (NOX) (Wang et al., 2014) In the same disease model, it was revealed that the H2S treatment can promote angiogenesis and thus improve the functional outcome after cerebral ischemia (Jang et al., 2014) In one of the latest studies, Wen and

team have reported that H2S improved the survival rate of hippocampal neurons thus reducing the learning and memory impairment in the rats with induced ischemic stroke H2S increased the phosphorylation of Akt while inhibited the phosphorylation of ASK1 and JNK3 (Wen et al., 2014)

1.2 Alzheimer’s disease

1.2.1 History

About a century ago, during a lecture at the 37th annual conference of German psychiatrists in Tubingen, a German neuropathologist and psychiatrist named Dr Aloysius "Alois" Alzheimer described ‘a particular disease of cerebral cortex’ of his patient Mrs Auguste Deter The patient had presented with the history of impaired memory, cognitive impairment, hallucinations, delusion, aphasia, disorientation and psychosocial incompetence A detailed post-mortem analysis of her brain revealed many peculiar findings such as atrophied brain and presence of plaques and neurofibrillary tangles Later, this presenile dementia, on suggestion of Dr Alzheimer’s boss Emil Kraepelin, became known as Alzheimer’s disease (AD) (Maurer et al., 1997) As of today, AD is the single most common cause of dementia in elderly population across the world (Reitz et al., 2011a) AD still has undetermined etiology and it’s definitive diagnosis can only be obtained post-mortem Currently, lots of efforts have been put in the development of drugs targeting the AD pathology

1.2.2 Epidemiology

In 2005, 24.2 million people worldwide had dementia The developed world i.e north America and western Europe showed the highest prevalence

of dementia It is estimated that these parts of the world along with China will

be home to around 55% of total affected population worldwide by 2040 The prevalence of dementia grows with the age The extensive Delphi consensus study done by Ferri et al revealed the growth from 1% in 60-64 years age group to about 30% in those of >85 years age group The incidence rate of AD and other dementias also increase exponentially with the age, mirroring the prevalence rate The worst affected is the seventh and eighth decades of life (Ferri et al., 2005, Reitz et al., 2011b, Reitz et al., 2011a)

Region

Consensus Dementia Prevalence

at age >60 years (%)

Est annual incidence of dementia (per 1000 individuals)

People with dementia aged >60 years in

2001 (millions)

Est increase

in proportion

of people with dementia from 2001-

2014 (%)

Western

Table 2 Prevalence and incidence of dementia in developed and developing regions Data

is reproduced from (Ferri et al., 2005) with some modifications

1.2.3 Risk factors

A risk factor is any attribute, characteristic or exposure of an individual that increases the likelihood of developing a disease or injury (WHO, 2014) Various risk factors have been found to be associated with dementia and AD Recent advances indicate dementia risk is modified by perinatal events, education status, nutritional intake, degree of physical activity, and cognitive and social engagement Several of these factors impact adult-onset vascular disorders such as stroke, hypertension, atherosclerotic disease, type 2 diabetes mellitus, hyperinsulinemia, hyperglycemia, dyslipidemia, hyperhomocysteinemia and obesity It is increasingly recognized that factors that increase cardiovascular disease or brain vascular pathology exacerbate the

onset or progression of late-onset dementias and AD (Kalaria, 2010, Reitz et al., 2011a)

1.2.4 Pathology

Classically, AD pathology is characterized by the formation and accumulation of misfolded proteins (plaques and tangles) in the brain Amyloid β and tau have been identified as main components of plaques and tangles respectively

1.2.4.1 Amyloid β

The formation of extracellular plaques is described by generally accepted amyloid cascade theory The theory states that extracellular plaques are primarily made up of Aβ, which is a 40 to 42 amino acids long peptide and

is generated by sequential proteolytic cleavage of the larger amyloid precursor protein (APP) (Selkoe, 1991) In non-amyloidogenic pathway, APP is cleaved within the Aβ domain by α- secretase, releasing soluble APP (sAPPα) extracellularly On the other hand, poorly soluble amyloidogenic Aβ is derived from sequential cleavage of APP by β- and γ-secretases (Cummings et al., 1998)

About 25 years ago, it was contemplated that certain mutations in APP gene would be detected in familial AD (Goate et al., 1989) Since then, 20 mis-sense mutations have been described in the literature (Goedert and Spillantini, 2006) The mutations in APP lead to increased production of Aβ and/or increased ratio of Aβ42 to Aβ40 However, it should be noted that APP

mutations don’t account for majority for familial AD cases The mutations in presenilin-1 (PS1) gene have proven to be mainly responsible for familial AD (Schellenberg et al., 1992, Sherrington et al., 1995) Mutations in presenilin-2 gene have also been found to initiate AD pathology (Rogaev et al., 1995) In fact, scientists have recognized around 150 mutations in presenilin genes (Goedert and Spillantini, 2006) Presenilins form the catalytic subunits of high-molecular weight complex of γ-secretase (De Strooper et al., 1998) PS1 mutations result into reduced γ-secretase activity (Citron et al., 1997) and increased proportion of Aβ42 in overall production of Aβ The pathological investigations done in preclinical cases with PS-1 mutations, Aβ42 deposition was found out to be an early event (Lippa et al., 1998) It is interesting to know that no mutations were detected in BACE1, which is involved in the rate-limiting step of β-secretase cleavage of APP in Aβ formation (Vassar et al., 1999) Overall, the findings on familial AD support amyloid cascade theory, which expounds that elevated Aβ42 levels initiate AD pathology, which set up the cascade of downstream events

1.2.4.2 Tau

Another major hallmark of AD related changes in the brain is intracellular development of neurofibrillary tangles (NFTs) NFTs are primarily made of paired helical filaments (PHF) The main component of the NFTs is tau, a microtubule related protein (MAP) (Grundke-Iqbal et al., 1986)

It provides structural stability to a cell by binding to microtubulin Tau protein accumulation results from its dissociation from the microtublin (Su et al., 1996) The reason behind this aggregation phenomenon is explained by the tau hypothesis (Su et al., 1996) Under physiological conditions, soluble tau

undergoes phosphorylation and dephosphorylation, forming insoluble aggregates Any imbalance results into elevated levels of abnormally hyperphoshorylated tau (P-tau 181, P-tau 199, P-tau 231, P-tau 396 and P-tau 404), which in turn sequesters normal tau and other MAPs (MAP1 and MAP2) (Blennow et al., 2007) PHF and tangle formation are direct results of aggregation of hyperphosphorylated tau The microtubules disassembly is another process, which runs parallel to the process of tangle formation Both the process result into dysfunctional neuronal and synaptic function (Blennow

et al., 2006) The amyloid cascade hypothesis states that elevated levels of Aβ can trigger the changes in tau protein culminating into formation of NFTs It has been shown that protein α-synuclein (core components of lewy body based pathologies, known as synucleinopathies), like tau is involved in microtubule assembly It serves as a binding for the tubulin Mutations in α-synuclein lead

to loss of this binding ability, resulting in tubulin and α-synuclein aggregation Properly functioning microtubules are important for normal neuronal and synaptic functions Any alteration in microtubule assembly may be key event

in neurodegenerative disease (Alim et al., 2004)

1.2.4.3 Studies done on animal models and AD patients

The studies conducted on P301L human tau mutation transgenic mouse model have shown the increased production of tangles (Van Dam and De Deyn, 2006) It supports the notion of the core symptoms of AD have neurobiological basis with the pathological accumulation of amyloid peptide and NFTs, often growing independently, and with different distribution pattern (Braak et al., 1999, Van Dam and De Deyn, 2006) The topographic progression of AD in accordance with time has been demonstrated in a study

on post mortem brains of AD patients The brain regions involved are the medial temporal cortex, hippocampus, and entorhinal cortex, anterior cingulate gyrus (as well as disruption of the neocortex) - whilst other areas are unaffected - prominently cerebral and cerebellar cortex This distribution or topographically predictive nature of aggregation is thought to be a V to VI stage process whereby the first three stages are preclinical, with symptomatic

or clinically diagnosable symptoms becoming prominent from stages III onwards The amnesia occurs due to the affected hippocampus producing early memory changes and ultimately the progression to the final stage, where the neocortex is affected (Braak and Braak, 1991, Braak et al., 1999) Moreover, it has been recently shown that tangle formation is an early event to amyloid deposition (Braak and Del Tredici, 2004, Schonheit et al., 2004) This

is contrast to amyloid cascade theory, which states that amyloid deposition leads to tangle formation (Hardy and Higgins, 1992) The novel creation of 3xTgAD APP Swedish mutation has supported this hypothesis (Oddo et al.,

2003, Van Dam and De Deyn, 2006) showing amyloid deposition, as an event preceding tangle formation Combination of the APP Swedish mutation and P301L models have yielded both sets of pathologies; with amyloid considered

a bit more pathologic in nature, and therefore more likely to cause dementia

1.2.4.4 Other etiopathological hypotheses

Oxidative Stress

Oxidative stress damages various biomolecules in an unregulated manner and is considered to be one of the hallmark pathological features of neurodegenerative diseases It is also believed that the plaques and tangles are the results of protective antioxidant defense mechanism, rather than being